DNA construct comprising a vector for expression of human cytoplasmic Cu/Zn superoxide dismutase in E. coli and method of use of same

ABSTRACT

Methods and compositions are provided for the production of human superoxide dismutase and a novel protocol for enhancing efficiency of expression. The gene encoding for human superoxide dismutase is isolated and inserted into a vector in conjunction with a synthetic linker which provides for enhanced efficiency in translation.  E. coli  strain D1210 (pSODX8) was deposited at the A.T.C.C. on Sep. 27, 1983 and given Accession No. 39453. Yeast strain 2150-2-3 (pC1/1GAPSOD) and  E. coli  strains D1210 (pSOD11) and D1210 (pS20R) were deposited at the A.T.C.C. on May 9, 1984, and given Accession Nos. 20708, 39679 and 39,680, respectively.

This is a Continuation of application Ser. No. 08/057,496, filed on May6, 1993 now abandoned; which in turn is Divisional of application Ser.No. 07/222,352, filed Jul. 20, 1988, now U.S. Pat. No. 5,252,476; whichin turn is a Rule 62 Continuation of application Ser. No. 06/931,920,filed Nov. 14, 1986, now abandoned; which in turn is a Rule 62Continuation of application Ser. No. 06/609,412, filed May 11, 1984, nowabandoned; which in turn is a Continuation-In-Part of application Ser.No. 06/538,607, filed Oct. 3, 1983, now abandoned.

DESCRIPTION OF SPECIFIC EMBODIMENTS

1. Field of the Invention

Superoxide dismutase (“SOD”) is in fact a variety of different enzymesfound in most living organisms. One function in mammals is to destroysuperoxide, a material naturally produced during phagocytosis. Thesuperoxide dismutases are characterized in families based on the metalassociated with the enzyme, where the metals vary amongst iron,manganese, copper and copper-zinc. Superoxide dismutase, e.g., frombovine liver, has found clinical use, particularly as ananti-inflammatory agent in mammals including humans. Other utilitiesinclude scavenging superoxide anions due to exposure of a host tovarious superoxide-inducing agents, e.g. radiation, paraquat, etc.;prophylaxis or therapy for certain degenerative diseases, e.g.,emphysema; food preservation; and the like.

It is therefore important that stable supplies of physiologicallyacceptable superoxide dismutase be made available, particularly for usein vivo as an anti-inflammatory agent or for other therapeutic purposes.For human application it would be preferable to employ the homologousenzyme to prevent or minimize possible immune response. By employingrecombinant DNA techniques, there is the opportunity to produce productsefficiently, which have the desired biological activities of superoxidedismutase, such as immunological and enzymatic activities.

2. Description of the Prior Art

The amino acid sequence of human erythrocyte Cu—Zn superoxide dismutaseis described in Jabusch et al., Biochemistry (1980) 19:2310-2316 andBarra et al., FEBS Letters (1980) 120:53-55. Bovine erythrocyte Cu—ZnSOD is described by Steinman et al., J. Biol. Chem. (1974)249:7326-7338. A SOD-1 cDNA clone is described by Lieman-Hurwitz et al.,Proc. Natl. Acad. Sci. USA (1982) 79:2808-2811. Concerning the effect onefficiency of translation of varying the untranslated region upstreamfrom the initiation codon, see Gheysen et al., Gene (1982) 17:55-63;Thummel et al., J. Virol. (1981) 37:683-697; and Matteucci and Heyneker,Nucl. Acids Res. (1983) 11:3113-3121.

SUMMARY OF THE INVENTION

Efficient production of polypeptides demonstrating the biologicalactivity of human Cu—Zn superoxide dismutase is demonstrated by thepreparation of cDNA of the major portion of the structural gene, linkingto a mixture of adapters providing for varying sequences extending fromthe ribosomal binding site to degenerate nucleotides in the codingregion, and insertion of the complete gene with its translationalsignals into an expression vector. Transformation of microorganismsresults in efficient production of a competent polypeptide demonstratingbiological activity of human Cu—Zn superoxide dismutase. The gene may befurther used for combining with secretory and processing signals forsecretion in an appropriate host.

Novel protocols are provided for enhancing expression of a polypeptideinvolving the use of mixtures of adapters having varying sequencesflanking the initiation site for translation, i.e., in the regionbetween the ribosomal binding site and translational initiation site andin the initial several 5′-codons of the polypeptide, where permitted byredundancy constraints of the genetic code.

Polypeptides acetylated at their N-terminus and methods for producingsuch acetylated polypeptides are also provided. By providing aparticular acetylation signal sequence at the 5′-end of the structuralgene for a desired polypeptide, the N-terminal amino acid will beacetylated when the gene is expressed in yeast. The acetylation signalsequence encodes for at least the first two N-terminal amino acids,where the first amino acid is either alanine or glycine, and the secondamino acid is a polar amino acid, usually being threonine, serine oraspartate. Acetylation of human superoxide dismutase produced in yeastis demonstrated when the first two amino acids are alanine andthreonine, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 indicates the DNA linker sequence and a flow diagram showing itsuse;

FIGS. 2 and 3 are flow diagrams indicating the preparation of plot5/SOD.

FIG. 4 indicates the sequence of both the coding strand of human SODcDNA (5′→3′) and the resultant translation product.

FIG. 5 illustrates the sequence of the isolated human SOD gene describedin the Experimental section hereinafter.

FIG. 6 is a restriction map of the isolated human SOD gene described inthe Experimental section hereinafter.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Methods and compositions are provided for the efficient expression ofpolypeptides demonstrating the biological activities of human Cu—Znsuperoxide dismutase (“hSOD”). The methods employ a DNA sequence (“hSODgene”) encoding a substantial portion of the amino acid sequence of hSODin conjunction with a translational initiation region optimized forexpression in the expression host. The hSOD gene is inserted into anappropriate vector for expression in a host, conveniently underconditions which allow for secretion, so as to harvest the SOD productfrom the extracellular medium.

Methods and compositions are also provided for the N-terminalacetylation of hSOD and other polypeptides. Hereinafter, acetylationrefers to addition at the amino terminus of polypeptides and proteins incontrast to modification of amino acid side chains, e.g., lysine, as isalso observed naturally. Acetylation of polypeptides and proteins isuseful for a number of reasons. Where the natural condition of thepolypeptide includes acetylation, as is the case for cytoplasmic hSOD,methods of expression which include acetylation provide a product havingthe desired natural structure and conformation. Where the product findspharmaceutical and/or in vitro or in vivo diagnostic use, the acetylatedmaterial will minimize or eliminate immunogenicity when administered toa host and/or exposed to biological samples. Also, acetylatedpolypeptides are likely to be more stable and resistant to degradationby proteases and thus enjoy a prolonged existance in the cell, blood orbody and tissue fluids.

The structural gene for hSOD or other polypeptide includes anacetylation signal sequence at the 5′-end thereof, which signal sequencecauses a yeast expression host to effect actylation. The acetylationsignal sequence encodes at least the first two N-terminal amino acids inthe polypeptide. The first amino acid will be either alanine, glycine orserine, while the second amino acid will be a polar or aromatic aminoacid, usually being threonine, serine, aspartate or phenylalanine.

The amino acids may be the natural N-terminal amino acids normallypresent in the polypeptide to be expressed. This is the case with hSODwhere the first two amino acids are alanine and threonine, respectively.Other naturally-acetylated proteins which may be expressed andacetylated in yeast include:

Protein Source Signal Sequence Cytochrome C Human, Rhesus GLY-ASPMonkey, Dog, Horse, etc. Cytochrome C Castor, Sesame, ALA-SER Mung-bean,etc. Glutamate Neurospora SER-ASN dehydrogenase Calmodulin Pig SER-ALAMyosin — SER-PHE (light chain A2) ADH Drosophila SER-PHE

The present invention is also useful for acetylating polypeptides andproteins which are not naturally acetylated. Acetylation may be achievedby joining the acetylation signal sequence to the 5′-end of thestructural gene for the polypeptide. The acetylation signal sequencewill encode for at least two amino acids (as described above), and mayencode up to ten or more amino acids, preferably fewer than five aminoacids. Fewer added amino acids is usually desirable to limitinterference with or loss of a desired activity of the polypeptide.Conveniently, the signal sequence may be synthesized and joined to thestructural gene using well known techniques.

As an alternative to adding the acetylation signal sequence to thestructural gene, it will sometimes be possible to modify the 5′-end ofthe structural gene to substitute one or both of the first two aminoacids of the polypeptide. Such modification may be accomplished by avariety of conventional methods. For example, the structural gene may berestricted near its 5′-end to remove a known number of nucleotides. Asynthetic oligonucleotide may then be joined to the cohesive endremaining after restriction. The oligonucleotide will restore andsubstitute the base pairs as necessary to provide the desiredacetylation signal sequence. Alternatively, site-specific mutagenesisemploying, e.g., phage M13, can be used to effect an appropriatemodification to the 5′-end of the structural gene.

In order to prepare hSOD, it is necessary to have a DNA sequence whichencodes for hSOD. One manner of achieving such sequence, is to clonecDNA from messenger RNA from cells which produce hSOD. Conveniently,human liver cells may be used for this purpose. After the cDNA iscloned, where the DNA coding sequence is unknown, but at least a partialamino acid sequence is known, one may then screen the cDNA with mixturesof probes having all of the possible variations of nucleotides encodingfor the particular series of amino acid residues. The choice of theresidues for which the sequence encodes is somewhat arbitrary, althoughthe residues chosen will usually be selected to minimize the number ofdifferent sequences which must be synthesized.

For hSOD, conveniently a DNA sequence encoding for at least the aminoacid residues 19 to 24 can be used, particularly a probe having at leastabout 15 bases and not more than about 20 bases, more conveniently about17 bases. One may then restriction enzyme digest the clones which appearto hybridize with the labeled probes, fractionate the DNA fragments andrepeat the hybridization, particularly by employing a second series ofprobes which hybridize to DNA sequences encoding for a different seriesof amino acid residues in hSOD. Conveniently, these amino acid residuesmay be 109 to 114. One or more clones may be found which are positive toboth probes and these may be used as a source for CDNA encoding for atleast a substantial proportion of hSOD.

Quite surprisingly, it was found that the amino acid sequences whichhave been published for hSOD differed in a significant number ofresidues from the amino acid sequence encoded for by the cDNA.Specifically, where the two published sequences differed (Jabusch etal., Biochemistry (1980) 19:2310-2316 and Barra et al., FEBS Letters(1980) 120:153-156), the correct assignments are: residue 11, aspartate;residue 17, isoleucine; residue 26, asparagine; residue 49, glutamate;residue 52, aspartate; residue 53, asparagine; residue 92, aspartate;residue 98, serine (see FIG. 4).

Because of the uncertainties of the effect on translation of theseparation between the ribosomal binding site and the translationalinitiation codon, normally AUG, the subject method provides a techniquefor varying the distance and nucleotides separating the ribosomalbinding site from the initiation codon. Usually, there are from about 6to 15, more usually about 6 to 12 nucleotides in the spacer between theribosomal binding site and initiation codon. As the base sequencedownstream from the initiation site may also affect translationefficiency, the subject method also provides for variation of nucleotidesequence (but not length) within the initial several 5′-codons of thepolypeptide as permitted by the redundancy constraints of the geneticcode. Such degeneracy may intend up to 4 codons, more usually 2 codons,downstream from the initiation site.

A plurality of linkers are predared where at least 2 nucleotides,usually at least 3 nucleotides, and not more than 10 nucleotides,usually not more than about 6 nucleotides, are varied to include membershaving each of the 4 nucleotides if within the spacer or 2, 3, or 4nucleotides as permitted by genetic code redundancy if within thestructural gene for the polypeptide. In addition, the linkers areprepared, having differing numbers of nucleotides, so as to provide agroup of linkers differing not only in the sequence, but also in length.The difference in length can be achieved by removal of portions of thesupport during the linker synthesis and, if appropriate, continuingsynthesis at a subsequent stage, so as to provide for linkers having agraduated number of sequence lengths. Usually, the mixture of linkerswill vary in length by at least one nucleotide and not more than over arange of six nucleotides, usually not more than four nucleotides.

This can be conveniently illustrated where the absent bases are at theterminus. After each stage, a portion of the support is removed and thesynthesis continued with the strands bound to the support, providing allfour nucleotides (dNTP) at each stage. These single strands will then behybridized to a single strand which is complementary in part, where thevariable region will be an overhang. Thus, one will achieve a graduatedseries of linkers having overhangs differing in both their nucleotidesequences and lengths. At an appropriate point during subsequenthybridization, ligation or cloning operations the overhang region(s) isfilled in to provide double-stranded material amenable to furthermanipulation. This is usually and preferably performed in vitro, e.g.,using the Klenow fragment of DNA polymerase I; alternatively, in certainconstructs the overhang could be cloned as a single strand with fillingin occurring in vivo in the transformed or transfected host.Hybridization to a complementary strand can be achieved by having a5′-sequence upstream from the variable nucleotide series which iscomplementary to a sequence present in the terminal sequence to whichthe linker is to be joined. The missing bases may then be filled invitro or in vivo.

The linkers include within their sequence, at least a portion of theregion between the ribosomal binding site and the initiation codon,preferably the nucleotides proximal to the initiation codon. The linkermay also include the initiation codon and portions of the structuralgene, the ribosomal binding site, and bases upstream from the ribosomalbinding site, which may or may not include transcriptional regulatorysequences.

Usually the linker will be at least about 5 bases, more usually at leastabout 20 bases, and usually not exceeding about 200 bases, more usuallynot exceeding about 100 bases. Where the linker is greater than about 35bases, it will usually be assembled by employing single strandedsequences of from about 10 to 35 bases, which have homology with only apart of a complementary strand, thus providing for complementaryoverlapping sequences with overhangs, so that the various single strandscan be hybridized, ligated and the degenerate and/or variable lengthoverhang filled in as indicated above to produce the desired linkerhaving cohesive and/or blunt ends.

Where the structural gene has a convenient restriction site, usually notmore than about 50 bases downstream from the initiation codon, afragment containing the structural gene may be restricted and joined toa complementary cohesive terminus of the linker or may be filled in toprovide a blunt-end terminus, which blunt end may be ligated to a bluntend of the linker. The linker is devised to ensure that the structuralgene is complete and in reading frame with the initiation codon.

As indicated, in preparing the linker, one provides that there are aseries of linkers which have a randomized series of nucleotides, thatis, each of the four possible nucleotides in the coding strand (subjectto the provision of genetic code limitations indicated above) and whichare graduated in size, lacking one or more of the nucleotides definingthe region intermediate or bridging the ribosomal binding site andinitiation codon. These linkers which are prepared from single strandsmay be joined to other single or double DNA strands to provide forextended linkers, which may include not only the ribosomal binding site,but bases upstream from the ribosomal binding site. Alternatively, thelinkers may be relatively small, beginning at a site internal to oradjacent to the ribosomal binding site and extending downstream to asite at the initiation codon or internal to the structural gene.

While the particular order of joining the various fragments to producethe constructs of this invention will usually not be critical,conveniently, the structural gene may be first joined to the linker.This DNA construct will include not only the structural gene, but alsothe ribosomal binding site and any additional nucleotides upstream fromthe ribosomal binding site. In addition, there will be substantialvariety in the nucleotides and numbers of nucleotides between theribosomal binding site and initiation codon. The subject DNA constructis inserted into an appropriate expression vector which has thenecessary transcriptional initiation regulatory sequences up-stream, aswell as transcriptional termination regulatory sequences downstream fromthe insertion site of the subject DNA construct. Thus, the linker willbe flanked at the 5′-end with transcriptional initiation regulatorysignal sequences and at the 3′-end with at least a portion of a codingregion and transcriptional and translational termination sequences. (5′-and 3′-intend the direction of transcription.)

After preparing the plasmid or viral DNA for introduction into anappropriate host (usually including at an appropriate stage in themanipulations filling in of the variable overhang region), the host istransformed or transfected, respectively, cloned, the clones streakedand individual clones selected for efficient expression by assaying forproduction of the desired product, e.g., hSOD. The number of clones tobe screened to determine the various levels of production of the productwill depend upon and be proportional to the degreee of lengthvariability and sequence degeneracy introduced into the syntheticlinker. As exemplified in the present embodiment, with 4 lengthvariables and 4-fold sequence degeneracy at each of 6 nucleotides in thelinker, the number of possible recombinant sequences is 5440. Usually atleast a few hundred, preferably several thousand or more, clones will bescreened. Screening can be efficiently performed using Western blots(antibody detection of product) of host cell colonies or viral plaquestransferred to filters of nitrocellulase or other suitable material.Alternatively, using electrophoresis and providing for a plurality oflanes, where each lane is an individual clone, an immediate and directcomparison can be made of which clones are most efficient in expressionby visualization of staining intensity, autoradiography or Westernblotting of the product band. This screen will usually be sufficient,although more quantitative immunoassays or enzyme assays can beemployed, as appropriate.

If desired, the construct can be transferred to a different host whichrecognizes the regulatory signals of the expression construct or theexpression construct modified by introduction at appropriate sites ofnecessary regulatory signals to provide for efficient expression in analternative host.

If desired, the hSOD gene may be joined to secretory leader andprocessing signals to provide for secretion and processing of the hSOD.Various secretory leader and processing signals have been described inthe literature. See for example, U.S. Pat. Nos. 4,336,336 and 4,338,397,as well as copending application Ser. Nos. 522,909, filed Aug. 12, 1983and 488,857, filed Apr. 26, 1983, the relevant portions of which areincorporated herein by reference.

Of particular interest as hosts are unicellular microorganism hosts,both prokaryotes and eukaryotes, such as bacteria, algae, fungi, etc. Inparticular, E. coli, B. subtilis, S. cerevisiae, Streptomyces,Neurospora may afford hosts.

A wide variety of vectors are available for use in unicellularmicroorganisms, the vectors being derived from plasmids and viruses. Thevectors may be single copy or low or high multicopy vectors. Vectors mayserve for cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers are present, which providefor different characteristics.

In addition to the markers, vectors will have a replication system andin the case of expression vectors, will usually include both theinitiation and termination transcriptional regulatory signals, such aspromoters, which may be single or multiple tandem promoters, an mRNAcapping sequence, a TATA box, enhancers, terminator, polyadenylationsequence, and one or more stop codons associated with the terminator.For translation, there will frequently be a ribosomal binding site aswell as one or more stop codons, although usually stop codons will beassociated with a structural gene. Alternatively, these regulatorysequences may be present on a fragment containing the structural gene,which is inserted into the vector.

Usually, there will be one or more restriction sites convenientlylocated for insertion of the structural gene into the expression vector.Once inserted, the expression vector containing the structural gene maybe introduced into an appropriate host and the host cloned providing forefficient expression of hSOD.

In some instances, specialized properties may be provided for thevector, such as temperature sensitivity of expression, operators oractivators for regulation of transcription, and the like. Of particularinterest is the ability to control transcription by exogenous means,such as temperature, inducers, corepressors, etc., where transcriptioncan be induced or repressed by an exogenous compound, usually organic.

Where the hSOD is made intracellularly, when the cell culture hasreached a high density, the cells may be isolated, conveniently bycentrifugation, lysed and the hSOD isolated by various techniques, suchas extraction, affinity chromatography, electrophoresis, dialysis, orcombinations thereof. Where the product is secreted, similar techniquesmay be employed with the nutrient medium, but the desired product willbe a substantially higher proportion of total protein in the nutrientmedium than in the cell lysate.

The hSOD which is formed has substantially the same amino acid sequenceas the naturally occurring human superoxide dismutase, usually differingby fewer than 5 amino acids, more usually differing by fewer than 2amino acids. The recombinant hSOD (“r-hSOD”) displays substantially thesame biological properties as naturally occurring hSOD. The biologicalproperties include immunological properties, where antibodies raised toauthentic hSOD cross-react with r-hSOD. Furthermore, in common bioassaysemployed for hSOD, the r-hSOD product demonstrates a substantialproportion, usually at least about 10%, preferably at least about 50%,more preferably at least about 80%, of the enzymatic activity of theauthentic hSOD, based on weight of protein. An illustrative assaytechnique is described by Marklund and Marklund, Eur. J. Biochem. (1974)47:469-474.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Molecular Cloning of hSOD cDNA

Total RNA was prepared from an adult human liver by the guanidiniumthiocyanate/lithium chloride method (Cathala et al., DNA (1983)2:329-335). polyA RNA was used to synthesize double-stranded cDNA(Maniatis et al., Molecular Cloning, 213-242, Cold Spring Harbor, 1982)and this was passed over a Sepharose CL4B column to enrich for cDNAs ofgreater than 350bp (Fiddes and Goodman, Nature (1979) 281:351-356). ThecDNA was inserted at the PstI site of plot4, a pBR322 derivative havingthe following sequence replacing the PstI-EcoRI site.

PstI    HinfI               AluI   1     GGTGAATCCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTC ACGTCCACTTAGGCATTAGTACCAGTATCGACAAAGGACACACTTTAACAATAGGCGAG     HphI                                   HindIIAluI  60ACAATTCCACACATTATACGAGCCGATGATTAATTGTCAACAGCTCATTTCAGAATATTTTGTTAAGGTGTGTAATATGCTCGGCTACTAATTAACAGTTGTCGAGTAAAGTCTTATAAA                     EcoRI 120 GCCAGAACCGTTATGATGCGGCGGTCTTGGCAATACTACGCCTTAA

The cDNA insertion employed the oligo-dG:dC tailing method (Maniatis etal., supra). E. coli strain D1210 was transformed with this mixture andtransformants selected on L-agar containing 10 μg/ml tetracycline(Kushner, S. R. (1978) In: Genetic Engineering, eds. Boyer, H. B. andNicosia, S., (Elsevier/North Holland, Amsterdam) p. 17). Plasmid DNAconstituting a liver cDNA library was prepared (Maniatis et al.,Molecular Cloning, pp. 86-94, Cold Spring Harbor 1982) directly fromapproximately 62,000 recombinant colonies plated at a density ofapproximately 3,000 colonies per 9 cm diameter Petri dish.

Isolation of r-hSOD Clones

Strain D1210 was retransformed with the liver cDNA library and about40,000 clones were grown on nine 14 cm diameter Petri dishes. Aftertransfer of the colonies to nitrocellulose paper and chloramphenicolamplification of plasmid DNA, the cells were lysed and the filtersprepared for hybridization (Ish-Horowicz and Burke, Nucleic AcidsResearch (1981) 9:2989-2998). Oligonucleotide probes were employed forscreening by hybridization, with the probes consisting ofenzymatically-radiolabeled, chemically-synthesized DNA moleculescomplementary to the mRNA encoding amino acid residues 19 to 24 of theprotein (Jabusch et al., supra.; Barra et al., supra.); the mixture hadthe following sequences:

3′ TTA AAA CTT GTT TTT CT 5′      G   G   C   C   C      

where all of the indicated possibilities for encoding the peptidesequence were prepared (32-fold degenerate).

The probes were labeled with ³²P to a specific activity of 1-3×10⁸cpm/μg and Millipore (0.45 μm) filtered before use. Filters wereprehybridized for 6 hrs at 30° C. in 4×SSC, 2×Denhardts's solution, 40mM sodium phosphate, pH 7.5, 300 μg/ml sonicated salmon testes DNA.Hybridization was for 20 hrs at 30° C. in the same solution containing2×10⁶ cpm/ml hSOD DNA probe (residues 19-24). Filters were washed in4×SSC, once for 15 min at r.t. and twice for 15 min at 30° C., blotteddry and autoradiographed with an intensifying screen for 24 hrs at −70°C.

Areas on the master plates that corresponded to duplicate positivesignals were picked into L-broth and plasmid DNA prepared by theminiscreen procedure (Maniatis et al., Molecular Cloning, 178, 368-369,Cold Spring Harbor 1982). This DNA was cut with PstI and subjected toSouthern blot analysis (Southern, J. Mol. Biol. (1975) 98:503-517)hybridizing initially with the previous labeled probes (amino acidresidues 19-24) and then with additional radiolabeled probes derivedfrom amino acid residues 109-114 and having the following sequences (allpossible variations, 72-fold degenerate) present as a mixture:

3′ CTA GTA ACA TAA TAA CC 5′      G   G   G   G   G                       T   T      

One plasmid pool (pSOD1) contained a CDNA insert of 520 bp thathybridized with both probes and after colony purification, plasmid DNAwas prepared from this clone and sequenced by the method of Maxam andGilbert (Proc. Nati. Acad. Sci. USA (1977) 74:560-564) with the resultsshown in FIG. 4. The hSOD cDNA clone pSOD1 constitutes the coding regionfor amino acids 10-153 of hSOD, a single translational stop codon and a3′ untranslated region. Therefore, in the expression vector construct,the base sequence of the region encoding amino acids 1-9 is derived fromthe published amino acid sequence of hSOD (Jabusch et al., supra; Barraet al., supra) and synthesized chemically as a part of the variablelinker segment (see below). Construction of Plasmid plot5—(See FIGS. 2and 3)

Plasmid plot1, containing a hybrid trp-lac (“tac”) promoter (DeBoer etal., Proc. Natl. Acad. Sci. USA (1983) 80:21-25) was constructed by gelisolating the 180 bp HgiA-TaqI fragment of ptrpL1 (Edman et al., Nature(1981) 291:503-506) and the 58 bp HpaII-EcoRI fragment from pKB268(Backman and Ptashne, Cell (1978) 13:65-71), and ligating thesefragments to pBR322 digested with PstI and EcoRI. The resulting plasmidwas used to transform strain D1210 and clones selected for tetracyclineresistance. Plasmid plot3 was constructed by gel isolating the 155 bpFnu4HI-EcoRI fragment of plot1 containing the tac promoter, with theFnu4HI site being made flush-ended using the Klenow fragment of DNApolymerase I (“pol I KX” or “pol. Klen.”), and the 18 bp EcoRI-PstIpolylinker fragment of πAN7 of the following sequence:

These fragments were ligated to gel purified pBR322 digested with EcoRI,flush-ended using pol I K, followed by digestion with PstI and gelpurified. This ligation mix was used to transform strain D1210,selecting on L-agar plates containing 10 μg/ml tetracycline.

Plasmid plot5 was made by first constructing a plasmid containing theπAN7 polylinker as an EcoRI-PvuII substitution in pBR322. To do this,plasmid πAN7 was digested with HindIII, made flush-ended by filling inwith pol I K and a synthetic, self-complementary, PvuII linker molecule(d(5′-CCAGCTGG-3′)) ligated to the above-modified plasmid πAN7. Afterdigestion with EcoRI and PvuII, the resultant 44 bp polylinker fragment(with 4-base overhangs) was gel isolated and cloned into pBR322 as anEcoRI-PvuII substitution.

Plasmid plot3 was digested with EcoRI and after phenol-chloroformextraction and ethanol precipitation, the protruding 5′-ends were madeflush-ended by treatment with S1 nuclease (Palmiter, Biochemistry (1974)13:3606-3615; Hallewell and Emtage, Gene (1980) 9:27-47). Afterphenol-chloroform extraction and ethanol precipitation, the DNA wasdigested with ClaI, made flush-ended by pol I K, and the 237 bp fragmentcontaining the tac promoter isolated by preparative polyacrylamide gelelectrophoresis. This flush-ended tac promoter fragment was theninserted at the PvuII site of the pBR322 polylinker plasmid (see FIG. 3)and clones obtained in which the tac promoter directed transcriptiontowards the β-lactamase gene of pBR322.

Construction of plot5 Derivatives Expressing r-hSOD

The synthetic DNA molecules F(26), C(16), B(31), D(11), E(13) and 4(24)shown in FIG. 1, were synthesized by the phosphoramidite method. Thesingle strand 4(24) was prepared by using all four bases, at each sitewhere X is indicated. Furthermore, silica was withdrawn from thesynthesis of the 24 mer, such that single-stranded 21 mers, 22 mers, and23 mers are obtained in addition to the 24 mers. After removal from thesilica support, the four mixtures are combined in appropriateproportions to provide for equimolar amounts of each of the possiblesingle strands. This mixture was treated as a single product in thesubsequent steps.

Molecules F(26), C(16), B(31) and D(11) were mixed together in equimolaramounts and 10 μg phosphorylated using T4 polynucleotide kinase. Afterphenol-ether extraction, the additional non-phosphorylated synthetic DNAmolecules 4(24) and E(13) were added, such that all fragments wereequimolar. The equimolar mixture contained 13 μg of DNA in 133 μl of0.3× kinase buffer.

After annealing by cooling at a uniform rate from 70° C. to 20° C. over60 min, the single strands were ligated together with T4 ligase in 200μl ligation mix at 14° C. for 4 hrs, phenol-chloroform extracted,ethanol precipitated and the 5′-ends of 4(24) and E(13) phosphorylatedusing T4 polynucleotide kinase (Maniatis et al., supra). Preparativepolyacrylamide gel electrophoresis was used to isolate the completelyligated 53 bp material having 5′- and 3′-overhangs.

The above purified fragment mixture was then ligated to the 460 bpTaqI-PstI segment of the hSOD cDNA as shown in FIG. 1. This segment wasitself constructed by isolating the 454 bp TaqI-AluI hSOD fragment,making it flush-ended pol I K using and inserting it into plotbetweenits EcoRI and SalI sites (see FIG. 3) which had been similarly madeflush-ended. After preparation of plasmid DNA from this recombinant, the460 bp TaI-PstI hSOD fragment was isolated by preparative polyacrylamidegel electrophoresis. After extraction and precipitation, the 515 bpfragment resulting from the joining of the synthetic fragment to the 460bp TagI-PstI hSOD fragment was filled in with pol I K (525-528 bp) andthen digested with SalI and the resulting 519-522 bp hSOD fragmentisolated by polyacrylamide gel electrophoresis. This fragment was theninserted into plot5 which had been digested with PvuII and SalI and thentreated with alkaline phosphatase. The resulting plasmids were used totransform strain D1210. Recombinants obtained after transformation ofstrain D1210 were selected on L-agar containing 100 μg/ml ampicillin togive a set of clones (designated plot5/SOD) with variable SODexpression.

r-hSOD Expression and plot5/SOD Plasmid Selection

For analysis of total E. coli proteins by SDS-polyacrylamide gelelectrophoresis, overnight cultures were diluted 30-fold into 1 ml ofL-broth and grown shaking at 37° C. for 90 min. to an O.D.₆₅₀ of about0.2. IPTG (isopropylthiogalactoside) was added to a final concentrationof 2 mM and the cultures incubated an additional 3 hrs. Aftercentrifugation, the cell pellet was resuspended in 50 μl of gel loadingbuffer (Laemmli, Nature (1970) 227:680-685) and lysed by repeating thefollowing procedure 3x: Freezing for 1 min., boiling for 2 min.,vortexing for 10 sec.

After electrophoresis resolution (Laemmli, supra) the protein bands werestained with Coomasie blue and the amount of SOD produced by each cloneestimated; these results were then confirmed using Western blots withantibody to authentic human SOD. Over three hundred clones were analyzedand exhibited levels of SOD expression varying from little or none toamounts estimated to be 5-10% of the total soluble cellular protein.Results for two of the over three hundred clones are presented in Table1, along with the particular sequence for DNA molecule 4(24) asdetermined by the method of Maxam and Gilbert, supra.

TABLE 1 Sequence and Levels of SOD Production in E. coli Sequence:Approximate Weight Clone 5′-XXXX ATG GCX ACX Percent of Total ProteinpSODx8 AACA  A  G 5% pSOD11 GTAT  T  G

hSOD assays were performed along the pyrogallol method (Marklund andMarklund, supra) The reaction mixtures employed 0.2mM pyrogallol inassay buffer and reaction rates were determined over a 5 min. periodusing a Hewlett-Packard 8450 spectrophotometer at 420 nm. Four differentassay samples were prepared: soluble E. coli extracts; authentic hSOD;and each of the prior samples pre-incubated with rabbit antibody toauthentic hSOD. Each sample was incubated in a cuvette for 1 min. at 25°C. before adding the pyrogallol and assaying at 25° C. The antibodysamples involved a preincubation of 10 min. at room temperature in assaybuffer with 5 μl of antibody. These conditions were found to besufficient to inactivate 100 ng of pure hSOD.

The following Table 2 indicates the results for one of the clonesexamined (pSOD×8):

TABLE 2 Enzymatic Activity of Human Cu—Zn SOD Produced in E. coli(strain D1210 (pSODX8)) Enzyme Preparation Units SOD/mg Protein pureHuman Cu—Zn SOD 15,384 pSODX8 protein extract 3,017 pSODX8 proteinextract 685 preincubated with rabbit anti-human SOD antibody plot5protein extract 470 plot5 protein extract 485 preincubated with rabbitanti-human SOD antibody

These data indicate that approximately 15% of the total soluble cellularprotein was hSOD (assuming that the pure human Cu—Zn SOD used as areference was fully active). Taken together with the electrophoreticdata (see above) indicating that 5-10% of total soluble cellular proteinmigrated as hSOD, it appears that a substantial fraction, probably amajority of the hSOD produced is active.

The correct sequence of the cloned gene was determined by the method ofMaxam and Gilbert, supra. In addition, the first twelve amino acids atthe N-terminus were determined by automated Edman degradation. Thedetected sequence of amino acids was as follows:

ALA-THR-LYS-ALA-VAL-(CYS)-VAL-LEU-LYS-GLY-ASP-GLY—

The first ALA residue detected was present at a molar concentrationapproximately equal to that of the input peptide indicating the absenceof a blocked amino terminus. The CYS residue was not detected by themethod of amino acid analysis used, but its presence was inferred fromthe nucleotide sequence.

Thus, the (N-formyl-) methionine was removed from the bacterialexpression product and the material had the correct amino acidsequences, i.e, identical to that reported for cytoplasmic hSOD residues1-10, but the N-terminal ALA residue was not acetylated. Furthermore,the polypeptide made in E. coli migrated more slowly than the naturalprotein in 1% agarose gel (pH 8.6) electrophoresis which detectsdifferences in charge (Corning Universal electrophoresis film, stainedaccording to Clausen, Immunochemical Technique, p. 530-531), alsoindicating lack of acetylation. In addition, analysis of trypticpeptides of both the bacterial hSOD polypeptide and the purified,authentic (acetylated) natural protein revealed that all trypticpeptides were identical, except the bacterial N-terminal peptide whichmigrated differently, i.e., with a charge expected for a peptide lackingthe N-acetyl group.

Expression in Yeast

For transfer of the r-hSOD gene to a yeast vector, the plot5/SOD plasmidclones were screened for an NcoI restriction site at the 5′-end of thecoding region. For those plasmids where the variable nucleotides present5′ to the ATG initiation codon are CC, the sequence CCATGG provides anNcoI site. Clones were screened, and one was selected and designatedphSOD.

The plasmid phSOD was digested with NcoI and SalI and a 550 bp fragmentobtained, which included 1 nucleotide untranslated at the 5′-terminusand the entire coding region for hSOD. pPGAP (a yeast expression vectorcarrying the GAP promoter, prepared as described below) was digestedwith NcoI and SalI followed by treatment with alkaline phosphatase, andthe SalI-NcoI fragment substituted for the NcoI-SalI fragment in pPGAPto provide pPGAPSOD. BamHI digestion of pPGAPSOD resulted in a 2 kbfragment which was gel isolated and inserted into the BamHI site ofpC1/1 and pC1/1 GAL4/370, to yield plasmids pC1/1GAPSOD andpC1/1GALGAPSOD, respectively.

Plasmid pC1/1 is a derivative of pJDB219 (Beggs, Nature (1978) 275:104)in which the region corresponding to bacterial plasmid pMB9 in pJDB219was replaced by pBR322 in pC1/1. For preparing an expression vectorhaving secretory and processing signals, see U.S. application Ser. No.522,909. Plasmid pC1/1GAL4/370, a regulatable yeast expression vectorcontaining the GAL1/GAL10 regulatory region (controlled by the GAL4 geneexpression product) is prepared as described below.

Plasmids pC1/1GAPSOD and pC1/1GALGAPSOD were transformed into yeaststrain 2150-2-3 (available from Lee Hartwell, University of Washington)as described previously (Hinnen et al. Proc. Natl. Acad. Sci. USA (1978)75:1929-1933), with the results of expression set forth in the followingTable 3.

TABLE 3 Expression of Human SOD in Yeast Strain 2150 SOD² Plasmid CarbonSource μg/mg protein pCl/1 g, L¹ 0 pCl/1GAPSOD g, L 148 pCl/1GALGAPSODg, L 0.4 gal 68 ¹All cultures grown in Minus Leucine media with 2%lactic acid, 3% glycerol with or without 2% galactose to late log orearly stationary phase. ²Determined by RIA.

hSOD levels were measured using a standard radioimmuno-assay withiodinated authentic hSOD as standard. Constitutive synthesis from theGAP promoter leads to very high levels of hSOD production, of the orderof 10-30% of the total cell protein. The induction with galactose worksalmost as well, yielding about 7% of the cell protein as hSOD.

When hSOD is produced at these high levels, it is usually necessary toprovide zinc and copper ion to the product protein as a prosthetic groupin order to recover full enzymatic, i.e., catalytic, activity, e.g., bydialysis against 1 mM solutions of both zinc and copper sulfate.Alternatively, zinc and/or copper ion may be included in the growthmedia; this method also provides a means of selecting for strainsproducing high levels of hSOD and/or avoiding the loss of plasmidvectors expressing hSOD in otherwise non-selective media.

Construction of pPGAP

pGAP1, a plasmid prepared by insertion of a HindIII fragment containingthe GAPDH gene GAP49 (Holland and Holland, J. Biol. Chem. (1979)254:5466-5474) inserted in the HindIII site of pBR322, was digested withHinfI and a 500 bp fragment isolated. The fragment was resected withBal31 to remove about 50 or 90 bp, followed by ligation with HindIIIlinkers and digestion with HindIII. pBR322 was digested with HindIII,followed by treatment with alkaline phosphatase and the about 450 or 410bp fragment inserted to provide pGAP128.

pGAP128 was digested with HindIII, the fragment made blunt-ended withthe Klenow fragment and dNTPs and the resulting 450 bp fragment isolatedby gel electrophoresis. This fragment was inserted into SmaI digestedplot5, which had been treated with alkaline phosphatase, to provideplasmid plot5pGAP128, which contained about −400 to +27 bp of the GAPDHpromoter and coding region.

Yeast expression vector pPGAP having a polyrestriction site linkerbetween the GAPDH terminator and short promoter region was prepared asfollows. Plasmid plot5pGAP128 was digested with BamHI and TaqI to yieldan approximately 390 bp BamHI-TagI fragment having the −400 to −26 bp ofthe GAPDH promoter. The BamHI-TaI fragment was ligated to a syntheticfragment containing −25 to −1 bp of the GAPDH promoter and severalrestriction sites including NcoI and having the following sequence:

CGA₂TA₃(CA)₃TA₃CA₃CACCATG₃A₂T₂CGT₂AG₂T₂AT₃(GT)₃AT₃GT₃GTGGTAC₃T₂A₂GCA₂TC₂AGCT

to provide a BamHI-SalI fragment, which was digested with BamHI and SalIand used to replace the BamHI-SalI fragment of BamHI-SalI digestedpBR322 treated with alkaline phosphatase. After ligation, the plasmidpGAPNRS was obtained which was digested with BamHI and SalI to provide a400 bp BamHI-SalI fragment which was gel isolated. This fragment wasligated to an about 900 bp SalI-BamHI fragment containing the GAPDHterminator region and a short segment of 3′ coding region and theresulting 1.4 kb BamHI-BamHI fragment digested with BamHI. TheSalI-BamHI GAPDH terminator fragment was obtained by SalI and BamHIdigestion of pGAP2, a plasmid prepared by insertion of an about 3.3 kbBamHI fragment containing the GAPDH gene GAP49 (Holland and Holland,supra) into the BamHI site of pBR322. Plasmids pGAP2 and pGAP1 wereobtained as follows: A yeast gene library was prepared by insertingfragments obtained after partial digestion of total yeast DNA withrestriction endonuclease Sau3A in lambda-phage Charon 28 (Blattner etal., Science (1977) 196:161-169). The phage library was screened withDNA complementary to the yeast GAPDH mRNA and the yeast GAPDH gene fromone of these clones was subcloned as either an about 3.3 kb BamHIfragment in the BamHI site of pBR322 (pGAP-2) or as an about 2.1 kbHindIII fragment in the HindIII site of pBR322 (pGAP-1).

pBR322 was digested with EcoRI and SalI, the termini blunt-ended andligated to BamHI linkers, followed by BamHI digestion and theBamHI-BamHI 3.8 kb fragment gel isolated, recircularized byself-ligation, cloned and designated pBRΔR1-Sal. The 1.4 kb BamHI-BamHIfragment was inserted into the BamHI-digested, alkaline phosphatasetreated pBRΔR1-Sal vector to provide the plasmid pPGAP of about 5.3 kbwith the orientation in the opposite direction of the amp^(r).

Construction of GAL Regulated Containing Plasmids.

Plasmid pLGSD5 is prepared as described in Guarente et al., (1982)supra. The plasmid was manipulated as follows: After restriction withXhoI, the overhangs were filled in with the Klenow fragment of DNApolymerase I (“Klenow fragment”), ligated with EcoRI linkers (GGAATTCC)and then completely digested with ECORI and Sau3A to provide a 370 bpfragment which was isolated by gel electrophoresis and included theintergenic sequence between GAL1 and GAL10 genes of yeast, and providesfor the GAL4 regulation sequence of the GAL1 and GAL10 genes.

This fragment was inserted into pBR322 which had been completelydigested with EcoRI and BamHI, followed by treatment with alkalinephosphatase to prevent oligomerization resulting in plasmid pBRGAL4.

Plasmid pBRGAL4 was completely digested with Sau3A, the overhangs filledin with the Klenow fragment, and the resulting blunt-ended fragmentligated with SalI linkers (CGTCGACG), followed by digestion with SalIand XhoI. The resulting 370 bp fragment was isolated by gelelectrophoresis. This fragment has the original 370 bp yeast GAL4regulator sequence with XhoI and SalI termini.

The fragment was then cloned in the plasmid plots. plotwas prepared byinserting the 40 bp polylinker fragment of the following sequence

into pBR322 as an EcoRI-PvuII substitution followed by insertion of thetrp-lac promoter (Russell and Bennett, Gene (1982) 20:231-245) into thePvuII site with transcription oriented toward the polylinker sequence.plot5 was completely digested with SalI, followed by treatment withalkaline phosphatase and the 370 bp fragment inserted into plot5 toprovide plasmid plot5GAL4/370. This plasmid was then completely digestedwith BamHI and SalI to reproduce the individual fragment extended by 6bp of the polylinker fragment. This fragment was then ligated intopC1/1, which had been completely digested with BamHI and SalI followedby treatment with alkaline phosphatase to prevent recircularization. Theresulting plasmid was designated pC1/1GAL4/370. The BamHI-SalI fragmentis located in the pBR322 portion of the vector pC1/1.

The phSOD polypeptide made in yeast was shown to be identical to thenatural human protein. Migration of hSOD made in yeast was identical tothe protein in both polyacrylamide gel electrophoresis (with and withoutsodium dodecyl sulfate) and in agarose gel electrophoresis (see above).Moreover, when highly purified yeast polypeptide was subjected to twelvecycles of Edman degradation, the sequence was the same as that reportedfor the human protein (residues 1-10) made in E. coli set forth above.The level of detection, however, was only 5 to 10% of the expected levelon a molar basis relative to protein input. This reduced detection levelindicated that the N-terminal amino acid was blocked, i.e., probablyacetylated. The result was confirmed by comparative analysis of trypticpeptides derived from yeast-produced hSOD and authentic acetylated humanmaterial which showed that all the expected trypic proteins wereidentical in the two samples including the N-terminal one, thusindicating the presence of acetylated N-terminal ALA in the yeastexpression product.

Isolation of the Human SOD Gene

To isolate the human SOD gene, a bacteriophage lambda libraryrepresentative of the human genome (R. Lawn et al. (1978) Cell15:1157-1174) was screened with a radiolabelled DNA probe made from thehuman SOD cDNA. One million phage plaques were screened, and 13positively hybridizing plaques were purified. Restriction endonucleaseanalysis of the phage DNAs indicated that there are at least 5 differentgenes, suggesting that there are other SOD related genes and geneproducts. One candidate for such a gene is the recently discoveredextracellular Cu/Zn SOD (S. Marklund, (1982) Proc. Natl. Acad. Sci. USA79:7634-7638). To distinguish the authentic cytoplasmic Cu/Zn SOD genefrom the related ones we used synthetic DNA probes5′-AATGCTTCCCCACACC-3′ and 5′-CTCAGTTAAAATGTCTGTTTG-3′ corresponding toamino acid residues 19-26 and nucleotides 193-213 3′ from the terminatorcodon in the 3′ untranslated region, respectively. Only one of the 13genomic DNAs hybridized with these probes, indicating that it is theauthentic human cytoplasmic SOD gene. This was confirmed by DNA sequenceanalysis of the N-terminal region, as shown in FIG. 5 where the aminoacid sequence determined by protein sequencing is confirmed. This alsoshows that no preprotein exists for SOD since an in-frame terminationcodon exists nine nucleotides 5′ from the initiator methionine codon. Asshown in FIG. 5, the human Cu/Zn SOD gene contains interveningsequences. The map of the SOD gene shown in FIG. 6 indicates that thereis more than one intervening sequence.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A DNA construct comprising an expression vector,wherein said expression vector comprises in downstream order oftranscription: (1) a promoter; (2) a ribosomal binding site; (3) aninitiation codon and a DNA spacer sequence positioned between theribosomal binding site and initiation codon; (4) a human cytoplasmicCu/Zn superoxide dismutase structural gene in reading frame with saidinitiation codon having a stop codon at the 3′-terminus of said genes,wherein said DNA spacer sequence, said initiation codon, and the firsttwo codons of said human cytoplasmic Cu/Zn superoxide dismutasestructural gene comprise the sequence: X_(n)X_(n)X_(n)X_(n)ATGGCXACXwherein each n is independently 1, and each X is independently A, C, T,or G; and (5) a transcription terminator, wherein said DNA construct,upon introduction into E. coli, causes the expression of active humancytoplasmic Cu/Zn superoxide dismutase.
 2. The DNA construct of claim 1,joined to a replication system for extrachromosomal replication andmaintenance.
 3. Thc DNA construct of claim 1, which is prepared by themethod comprising: determining the optimum distance for expressionbetween said ribosomal binding sito and said initiation codon bypreparing a variety of DNA spacers between said ribosomal binding sitoand said initiation codon varying the length and composition, andselecting for optimized expression.
 4. The DNA construct of claim 1,which further comprises an operator in functional relationship with saidpromoter.
 5. The DNA construct of claim 1, wherein said promoter is ahybrid trp-lac promoter.
 6. The DNA construct of claim 4, wherein saidpromoter is a hybrid trp-lac promoter.
 7. The DNA construct of claim 1,wherein the four nucleotides 5′ to said initiation codon in said DNAspacer are selected from the group consisting of5′-AACA-3′ and5′-GTAT-3′.
 8. A method for preparing a polypeptide comprising humancytoplasmic Cu/Zn superoxide dismutase, said method comprising: growingan E. Coli containing a DNA construct according to claim 1 in a nutrientmedium, wherein said DNA construct causes the expression of active humancytoplasmic Cu/Zn superoxide dismutase; and isolating the humancytoplasmic Cu/Zn superoxide dismutase expressed from said DNAconstruct.